Leveraging Casein's Binding Power: Synergistic Experimental, Statistical, and Docking Insights into Methylene Blue Removal

Abstract Casein was used in this study to retain methylene blue dye in aqueous solutions. This study was described after characterizing casein using pHpzc, FTIR, ATG, and XRD analysis. FTIR spectra revealed characteristic bands of hydroxyl and amide groups confirming the protein nature of casein, along with methylene vibrations associated with aliphatic side chains. The hydroxyl (─OH), carbonyl (C═O), and amide (─CONH─) functional groups acted as the main active sites involved in dye binding. XRD patterns revealed a semi‐crystalline structure composed of α and β crystal forms, while TGA confirmed the good thermal stability of casein. The parameters influencing the adsorption are: contact time, initial concentration of the dye, pH, stirring speed, and temperature. Equilibrium was reached after 30 min with a discoloration percentage of 81.24%. The modeling of experimental results indicated that the adsorption process was exothermic, spontaneous and followed a pseudo‐second‐order kinetic model. For the optimization process, statistical techniques and response surface methodology (RSM) were applied, generating 32 experimental runs that identified four significant parameters: pH, dye concentration, temperature, and stirring speed. Docking simulations revealed that methylene blue binds within the hydrophobic core of β‐casein through two hydrogen bonds and two van der Waals interactions.